This work has the following general objectives concerning the unique broad host range capability of the Inc P-1 group of plasmids: (1) to identify and understand the gene functions needed for replication and maintenance; and (2) to determine the mechanisms of control that affect the expression of these genes in different hosts. One plasmid of this group, RK2, will be mapped intensively with a variety of in vivo and in vitro genetic methods. The genes which have been identified on this plasmid are known to function in host cell operating between these genes, several genetic fusions will be constructed between selected genes of RK2 and components of the lac operon. This will be done by cloning RK2 regulatory regions into sites preceding the structural gene for beta-galactosidase. Expression of this enzyme will then be monitored as an indicator of activity occurring at the RK2 control sequences. RK2 genes will also be placed under control of the lac promoter-operator region. These genetic fusions will permit the selection of mutants that would not otherwise be attainable by conventional mutagenesis of RK2 genes. A protocol has been devised that will permit mutations to be transferred from small easily-manipulable cloned fragments back into the parental RK2 to replace the wild-type allele. These mutant RK2 plasmids will be tested for effects on host range.